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Antibody against rat postacrosome reaction sperm and utilization thereof

a postacrosome and antibody technology, applied in the field of monoclonal antibodies, can solve the problems of time and cost, difficulty in establishing the method of fertility evaluation, and difficulty in obtaining the effect of sperm,

Inactive Publication Date: 2005-01-18
FUSO PHARMA INDS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is about developing a method for evaluating the fertility of spermatozoa quickly and accurately, using monoclonal antibodies specific to non-human animal spermatozoa. This method can be extrapolated to humans and can be used in reproductive and developmental toxicity studies. The method involves testing for the occurrence of the acrosome reaction, which is an essential condition for fertilization, using monoclonal antibodies produced using rats. The invention can also be used to screen materials that affect fertility and to evaluate the effect of drugs on the acrosome reaction of spermatozoa.

Problems solved by technology

This means that a method for evaluation of fertility has not yet been established.
These procedures, however, require skillful technique and troublesome operation as well as expensive equipment.
This necessitates time and cost.
Even though many points such as the number of spermatozoon, mobility and morphology, which appear to have some influence on fertilization, are examined, it is impossible to determine the exact fertility so long as the presence of acrosome reaction as an essential factor for fertilization cannot be confirmed.

Method used

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  • Antibody against rat postacrosome reaction sperm and utilization thereof
  • Antibody against rat postacrosome reaction sperm and utilization thereof
  • Antibody against rat postacrosome reaction sperm and utilization thereof

Examples

Experimental program
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Effect test

example 1

Method for Collecting Immunogens

Wistar male rats (age in 14 weeks) were killed by exsanguination under anesthesia with ether, from which the cauda epididymides were removed and immediately placed in a Petri dish containing 10 mL of m-KRB (modified Krebs-Ringer bicarbonate medium) containing 1% BSA (bovine serum albumin; Bayer) warmed at 37° C. (1 plate per cauda epididymis). Cleavages were made at 2-3 parts of the cauda epididymis, which was gently stirred and allowed to stand for about 5 minutes to release spermatozoa. The spermatozoa suspension is moved into a 50 mL tube, into which a calcium ionophore A23187 (Sigma) was added thereto at a final concentration of 10 μM and incubated for about 15 hours in a CO2 incubator. After terminating incubation, the treated spermatozoa were washed twice with PBS(−), then adjusted at a predetermined concentration, and preserved at −20° C.

example 2

Immunization of Mice

In Jcl female mice (age in 6 weeks), the first day of immunization was regarded as 0 day, and booster immunization was made on the 14th, 21st and 28th days. For immunization, the above-treated rat spermatozoa were administered subcutaneously in the back of the mice at a dose of about 1×107 per shot as an equal mixture with Freund's complete adjuvant and as an equal mixture with Freund's incomplete adjuvant, respectively, on day 0 and on the 14th day. On the 21st and 28th days, only spermatozoa were administered intraperitoneally.

example 3

Method for Preparation of Hybridoma

Three days after the final immunization, the spleen was removed from the immunized mice and used in cell fusion.

A suspension of splenic cells prepared on an RPMI1640 medium (GIBCO) was mixed with SP-2 cells (murine myeloma cells) at a ratio of about 4:1, permitted to undergo cell-fusion using polyethylene glycol 2000 (Sigma) and then washed with an RPMI1640 medium. Thereafter, the number of cells was adjusted to be about 2×106 cells / mL by dilution with an RPMI1640 medium containing 15% FCS (fetal calf serum; Mitsubishi Chemical), of which 100 μL each was distributed into each well of 96-well microplates, with 6 plates per mouse. The next day, 100 μL of HAT medium (hypoxanthine, aminopterin, thymidine, 15% FCS-containing RPMI1640 medium) was added to each well. On the 2nd, 3rd, 5th and 8th days, half of the medium was replaced with a HAT medium, and on the 10th and 13th days, half of the medium was replaced with an HT medium (hypoxanthine, thymidine...

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Abstract

An antibody biding specifically to rat's acrosome reacted sperm is produced and hybridomas (FARS-91 and FARS-92 strains) capable of stably proliferating are obtained by fusing mouse spleen cells having a high antibody titer against rat's acrosome reacted sperm with mouse-origin myeloma cells and screening fused cells reacting strongly with rat's acrosome reacted sperm. From these hybridomas, monoclonal antibodies selectively binding to rat's acrosome reacted sperm can be obtained. Thus, a diagnostic method for evaluating fertility of rat's spermatozoa is presented.

Description

TECHNICAL FIELDThe present invention relates to monoclonal antibodies selectively binding to rat acrosome reacted sperm, hybridomas producing said monoclonal antibodies, and a method for evaluating fertility of rat spermatozoa using said monoclonal antibodies. The invention also relates to a method for screening materials effecting on rat fertility, characterized in using said method for evaluating fertility.BACKGROUND ARTIt is now obligated to do reproductive and developmental toxicity studies in studies relating to safety of a drug on the occasion of an application of manufacturing approval for the drug. The reproductive and developmental toxicity studies mean that animal experiments are conducted to obtain information on whether the application of a drug to living bodies could possibly induce some adverse effect in the course of reproduction and development. The experimental results can be extrapolated to humans and utilized in the evaluation of safety (risk) of the drug to repro...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C07K16/18G01N33/577G01N33/68C12N5/20C12N15/06C12P21/08
CPCC07K16/18G01N33/689Y10S435/806Y10S435/975G01N2800/367
Inventor KUWAHARA, YOSHIHIROHASEGAWA, MICHINORIISAKA, KIYOTSUGUARAKI, HIROMASA
Owner FUSO PHARMA INDS
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